Small RNA is an important regulatory molecule with a length of 18-30nt, mainly including miRNA, siRNA and piRNA. It plays an important role in the regulation of gene expression, individual development, metabolism and disease. Small RNA sequencing uses Illumina sequencing platform to study all known small RNAs in a specific tissue of a species in a specific state. It can also find new or unique small RNAs of the species and predict their target genes. It provides a powerful tool for studying the function of small RNA and gene regulation mechanism of this species, and has been widely used in animal, plant researches and medical fields.

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Most small RNA experiments focus on different comparative groups comparison analysis, such as control vs case. If the space-time factors such as different growth and development stages or the whole process of disease occurrence and development are considered, multiple comparisons will be analyzed to obtain the significantly different miRNAs. Then target genes of those miRNAs are predicted and their enrichment analysis are carried out. At least 6 biological repeats are recommended for small RNA sequencing.